Cultured microorganisms produce an extraordinary array of structurally diverse and useful organic compounds. Molecular diversity measurements of microbial communities show that cultured microflora represent only a small fraction of the microbial diversity present in any natural environment. Currently unculturable microorganisms undoubtedly produce potentially valuable compounds and general methods to access these compounds are needed. An approach to study the natural products produced by uncultured microorganisms by introducing large fragments of DNA extracted directly from environmental samples (eDNA) into E. coli is described in co-pending parent application Ser. No. 09/558,712, filed Apr. 26, 2000. Heterologous expression of eDNA in an easily cultured host will provide access to the genomes of many uncultured microorganisms while bypassing the need to culture these organisms.
Natural products have traditionally been a rich source of small organic compounds that have found multiple uses in our daily lives. From the earliest known dyes and intoxicants to the most recent anti-neoplastic agents, natural products have been used by all human societies. The large collection of organic compounds that have been characterized from microbial sources is now known to have arisen from only a small sample of the earth's microbial diversity. Nucleic acids-based studies suggest that only a tiny fraction of any natural microbial community is cultured using standard microbiological methods. Uncultured bacteria have now been identified in a number of different environments including marine (1), gut (2, 3), hot springs (4), and soil (5, 6, 7). Uncultured microorganisms, therefore, represent one of the largest remaining unstudied pools of biodiversity. However, because these organisms are resistant to culturing, the natural products they produce remain inaccessible.
Using cloning and heterologous expression of large fragments of microbial DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means to access this previously untapped biodiversity. This approach has been termed “cloning the metagenome” to refer to accessing the collective genomes of the organisms in an environment (8). With the advent of bacterial cloning vectors such as the bacterial artificial chromosome (BAC), a vector that faithfully replicates very large fragments of DNA (9), it is now possible to clone and express in easily cultured hosts eDNA that codes for natural products produced by uncultured microorganisms. In short, direct cloning of eDNA provides a means to access the biosynthetic capacity of the genomes of heretofor uncultured microorganisms.
A metagenomics approach was first used to study the uncultured microflora present in soil samples. As with all other natural environments studied to date, the diversity of uncultured microorganisms in soil far exceeds that of cultured microorganisms. DNA-DNA reassociation kinetics and light microscopy analysis of soil samples indicate that cultured microorganisms represent less than 1% of the actual microbial diversity that exists in soil (10, 11). Analyzing small subunit rRNA gene sequences cloned from PCR-amplified soil DNA indicates that the uncultured microbial diversity in soil is much larger and more diverse than previously believed (5, 6, 7, 12). In addition to known examples of Archaea, Bacteria and Eukarya, analysis of small subunit rRNA gene sequences from soil microflora has consistently identified many sequences from previously uncharacterized microbial taxa. A recent analysis showed that nearly one third of the major divisions of phyla within the domain Bacteria are known only as environmental rRNA sequences (containing no cultivated members) and many of the well known divisions contain a wide diversity of as-yet-uncultivated members (13). Although there appears to be no easy way to culture this large collection of unstudied microorganisms, it is possible to isolate large fragments of microbial DNA directly from soil samples in high yields (>1 μg of DNA/gram of soil) (14, 15). The molecular diversity data from soil, together with the ease with which large eDNA can be harvested from soil samples, makes soil an ideal candidate for this new approach to the discovery of natural products.
Described herein are triaryl cationic antibiotics that are produced at elevated levels by a member of this library, pharmaceutical compositions containing the antibiotics, and methods of treating microbial infection using the pharmaceutical composition. The triaryl antibioutics were initially generated using a 25,000-member BAC library of eDNA extracted from soil (15; co-pending and co-owned U.S. patent application Ser. No. 09/558,712, filed Apr. 26, 2000), and subsequently synthesized de novo.